Unlike reverse transcriptase and protease, HIV integrase is difficult to study as an isolated enzyme because it functions in vivo in the context of the preintegration complex (PIC), a particle composed of both viral and host cell-derived components. In resting CD4+ T cells, this complex was found to be in an inactive form termed a ?pro-preintegration complex? or ?pro-PIC,? which is hypothesized to be the physical embodiment of preintegration latency. Cell stimulation is needed to convert the pro-PIC to an integration-competent PIC. Little is known about the molecular nature of the conversion process, and the exact composition of the PIC remains unknown. This project will address these issues from two opposite faces of the problem: on the host?s side, where immunological processes occur which trigger the formation of the active PIC; and on the virus?s side, where molecular changes convert the pro-PIC into the PIC. The four specific aims are: (1) To characterize the cellular stimuli that affect PIC activity in CD4+ T cells and macrophages; (2) To develop purification methods for pro-PICs and PICs from cells infected in vitro and to use them to establish the existence of pro-PICs and PICs in vivo in samples taken from HIV-infected individuals; (3) To define the structure and composition of pro-PICs and PICs using DNA footprinting, antibodies, and mass spectrometry; and (4) To screen PICs with a library of reactive compounds to find a putative new category of integration inhibitors which block PIC activity without directly inhibiting integrase. Taken together, these studies will clarify the linkage between the immunological processes that stimulate HIV replication and the molecular changes that they effect upon pro-PICs and PICs. New tools and concepts will be gained, and precise molecular targets for anti-HIV drug design will come into focus.